PATHWAYS OF TESTOSTERONE SYNTHESIS IN DECAPSULATED TESTES OF MICE

1976 ◽  
Vol 81 (1) ◽  
pp. 170-184 ◽  
Author(s):  
B. de la Torre ◽  
G. Benagiano ◽  
E. Diczfalusy
Keyword(s):  
Leydig Cell ◽  
Incubation Medium ◽  
Sodium Acetate ◽  
De Novo ◽  
Acetate Incorporation ◽  
Dehydroepiandrosterone Sulphate ◽  
Carbon 14 ◽  
Adult Mice ◽  
17 Hydroxyprogesterone ◽  
Testosterone Synthesis

ABSTRACT In order to study the temporal relations in the biogenesis of testosterone, decapsulated testes of adult mice were incubated with carbon-14-labelled sodium acetate and attempts were made to isolate the most likely intermediates. Considerable quantities of radiochemically homogeneous squalene, lanosterol, cholesterol, testosterone and androstenedione, but no pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, pregnenolone sulphate or dehydroepiandrosterone sulphate were isolated. The same pattern of incorporation was found when gradually increasing amounts of non-labelled pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate or testosterone were added to the system as "trapping agents" or when Leydig cell preparations rather than decapsulated testes were used. The presence of 10 mIU of HCG greatly enhanced the de novo formation of testosterone, androstenedione and 5α-dihydrotestosterone but did not change the pattern of acetate incorporation. Radioimmunoassays of the incubation medium with or without added HCG, and carried out at different periods of time indicated the presence of gradually increasing amounts of testosterone and androstenedione together with some 5α-dihydrotestosterone, whereas only trace amounts of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and dehydroepiandrosterone were present. An analysis of the incubated testes revealed that the addition of HCG significantly enhanced the content of testosterone, androstenedione and 5α-dihydrotestosterone. Little or no increase was observed as far as pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone or dehydroepiandrosterone were concerned. It is concluded that decapsulated testes of mice synthesize de novo testosterone from sodium acetate under conditions in which the formation of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnenolone sulphate and 17-hydroxypregnenolone sulphate cannot be demonstrated.

QUANTITATIVE ASSESSMENT OF THE DE NOVO STEROL AND STEROID SYNTHESIS IN THE HUMAN FOETO-PLACENTAL UNIT

1971 ◽  
Vol 66 (4) ◽  
pp. 666-678 ◽  
Author(s):  
D. F. Archer ◽  
R. S. Mathur ◽  
N. Wiqvist ◽  
E. Diczfalusy
Keyword(s):  
Sodium Acetate ◽  
Quantitative Assessment ◽  
De Novo ◽  
Isolation Procedure ◽  
Predominant Formation ◽  
Human Foetus ◽  
Steroid Synthesis ◽  
Internal Standards ◽  
Carbon 14 ◽  
Hydroxy Progesterone

ABSTRACT The quantitative significance of the de novo synthesis of Δ5- and Δ4-steroids was assessed in the midgestation human foetus. Two midgestation foetuses and two complete foeto-placental units were perfused at 36°C for 120 minutes with 3.0 and 6.0 mCi of [14C] sodium acetate, respectively, and radiochemically homogeneous carbon-14 labelled steroids were isolated from the adrenals, livers and perfusates. Losses throughout the isolation procedure were monitored in all tissues of all experiments by the recovery of tritium labelled internal standards. In the four experiments, a total of 18.0 mCi of [14C] sodium acetate was perfused and a total of 0.17 μCi of pregnenolone, 2.65 μCi of pregnenolone sulphate, 1.07 μCi of dehydroepiandrosterone and 8.64 μCi of dehydroepiandrosterone sulphate were isolated. A total of 12.53 μCi were isolated in the form of these four compounds; 10.13 μCi of this was present in the perfusates. In all, 0.07% of the administered [14C]-sodium acetate was converted to the four Δ5-steroids studied and 0.048% to dehydroepiandrosterone sulphate. Progesterone was isolated from the perfusates and liver extracts, and 17α-hydroxy-progesterone, androstenedione and testosterone from the perfusates. In the four experiments, a total of 0.045 μCi of carbon-14 labelled Δ4-steroids were isolated, predominantly from the perfusates. In all, less than 0.0003% of the perfused [14C] sodium acetate was converted to Δ4-steroids. Thus, almost 300 times more Δ5- than Δ4-steroids were formed. It is concluded that the extensive de novo steroidogenetic processes taking place in the midgestation human foetus lead to the predominant formation of Δ5-steroid sulphates.

IN VITRO STEROL AND STEROID BIOGENESIS BY A FEMINIZING ADRENOCORTICAL CARCINOMA

1973 ◽  
Vol 73 (3) ◽  
pp. 518-530 ◽  
Author(s):  
R. S. Mathur ◽  
H. O. Williamson ◽  
L. O. Moody ◽  
E. Diczfalusy
Keyword(s):  
Adrenocortical Carcinoma ◽  
Sodium Acetate ◽  
De Novo ◽  
Isotopic Ratio ◽  
Menopausal Woman ◽  
Isotopic Ratios ◽  
Carbon 14 ◽  
In Parenthesis ◽  
Post Menopausal

ABSTRACT A post-menopausal woman was found to excrete elevated amounts of urinary oestrogens. An adrenal carcinoma was removed and slices incubated in phosphate buffer, pH 7.4 with [14C] sodium acetate in combination with either [7α-3H] cholesterol, [7α-3H]dehydroepiandrosterone or [7α-3H]androstenedione. Radiochemically homogeneous tritium labelled oestrone, 17β-oestradiol and oestriol were isolated in each of the three experiments. Carbon-14 activity was incorporated into oestrone in each of the three experiments and into oestriol in one case only. No carbon-14 activity was associated with 17β-oestradiol isolated from any of the three incubations. Oestrone was found to be the predominant oestrogen synthesized in all experiments. When [14C]sodium acetate and [7α-3H]cholesterol (3H/14C ratio = 0.61) were used as substrates, the following compounds were isolated in a radiochemically homogeneous form (the isotopic ratio in each being shown in parenthesis): cholesterol (13.0), cholesterol sulphate (1.6), pregnenolone (1.0), pregnenolone sulphate (0.2), dehydroepiandrosterone (3.2), dehydroepiandrosterone sulphate (1.2), Δ5-androstenediol (3.1), Δ5-androstenediol sulphate (isolated as the unconjugated compound following solvolysis of the "disulphate" fraction (0.6), testosterone (1.1), testosterone sulphate (0.6), and oestrone (1.3). It is concluded that the adrenocortical carcinoma studied was capable of synthesizing oestrone, oestriol and cholesterol sulphate de novo from acetate, and that the steroidogenic processes led to the predominant formation of Δ5-steroid sulphates. Furthermore, a comparison of the isotopic ratios in the various compounds isolated suggests that a number of unusual transformations may have occurred during steroidogenesis by the tumour cells in vitro.

scholarly journals Currently available murine Leydig cell lines can be applied to study early steps of steroidogenesis but not testosterone synthesis

Heliyon ◽  
2018 ◽  
Vol 4 (2) ◽  
pp. e00527 ◽  
Author(s):  
Roger T. Engeli ◽  
Cornelia Fürstenberger ◽  
Denise V. Kratschmar ◽  
Alex Odermatt
Keyword(s):  
Cell Lines ◽  
Leydig Cell ◽  
Testosterone Synthesis

METABOLISM OF DEHYDROEPIANDROSTERONE SULPHATE AND OESTRONE SULPHATE FOLLOWING IN SITU PLACENTAL PERFUSION AT MIDPREGNANCY

1971 ◽  
Vol 66 (4) ◽  
pp. 637-647 ◽  
Author(s):  
J. Schwers ◽  
T. Vancrombreucq ◽  
M. Govaerts ◽  
G. Eriksson ◽  
E. Diczfalusy
Keyword(s):  
Radioactive Material ◽  
Dehydroepiandrosterone Sulphate ◽  
Placental Perfusion ◽  
Carbon 14 ◽  
Maternal Urine ◽  
Oestrone Sulphate ◽  
Unchanged Form ◽  
Urine Specimens

ABSTRACT Two midgestation placentas were perfused in situ with a combination of [7α-3H] dehydroepiandrosterone sulphate and [4-14C] oestrone sulphate and metabolites were isolated from the placentas, perfusates and maternal urine specimens. Approximately 70 per cent of the perfused radioactive material was recovered from these three sources. The bulk of the administered radioactive material was recovered in an unchanged form from the perfusates; some 2–4 per cent was excreted in the urine and less than 0.5% was found in the placentas. The tritium to carbon-14 ratio of the unconjugated material isolated from the perfusates and placentas was higher, and that of the conjugated material recovered from the same sources was lower than the ratio of the administered material. In addition, more tritium than carbon-14 labelled material was present in the urine. Approximately 2 per cent of the perfused dehydroepiandrosterone sulphate was recovered in the form of phenolic steroids, mostly from the urine. From this source double labelled oestrone, oestriol, 16α-hydroxy-oestrone and 16-epioestriol were isolated. The tritium to carbon-14 ratio of all oestrogens isolated from the urine was higher than that of the perfused material. From the urine specimens 10 to 15 times more double labelled oestriol than oestrone was isolated.

Abstract P477: A Novel SMYD1 Variant (c.302A>G) Leads To Dilated Cardiomyopathy And Biventricular Heart Failure.

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Marta Szulik ◽  
Miguel Reyes-Mugica ◽  
Daniel F Marker ◽  
Lina Ghaloul-Gonzalez ◽  
Sarah Franklin
Keyword(s):  
Heart Failure ◽  
Skeletal Muscle ◽  
Cardiac Tissue ◽  
De Novo ◽  
Cardiac Development ◽  
Left Ventricular ◽  
Histopathological Analysis ◽  
Loss Of Function ◽  
Adult Mice ◽  
Heterozygous Variant

The lysine methyltransferase SMYD1 was first identified in mice and shown to be important for embryonic cardiac development. Subsequently, we reported the first analysis of SMYD1 in adult myocardium and demonstrated that cardiomyocyte-specific loss of SMYD1 lead to progressive cardiac hypertrophy and heart failure, and showed that this enzyme is necessary to maintain metabolic homeostasis through transcriptional regulation of mitochondrial energetics in adult mice. While SMYD1 has been the subject of several additional studies in zebrafish and mice, since it was first identified, only in the last few years have human patients been identified with variants in the SMYD1 gene thought to be responsible for their cardiomyopathies. Specifically, two patients have been identified to date, the first patient displaying hypertrophic cardiomyopathy had a de novo heterozygous variant (c.814T>C) and the second patient with left ventricular non-compaction cardiomyopathy and arrhythmias had a truncating heterozygous variant (c.675delA). Here we report a third patient with biventricular heart failure containing a homozygous variant (c.302A>G; p.Asn101S) in the SMYD1 gene which was identified by a whole exome sequencing. Our histopathological analysis of cardiac tissue and skeletal muscle from the proband showed abnormalities in myofibrillar organization in both cardiac and skeletal muscle suggesting that SMYD1 is necessary for sarcomere assembly and organization. In addition, we observe markedly abnormal myocardium with extensive fibrosis and multifocal calcification, and our ultrastructural (EM) analysis revealed presence of abnormal mitochondria with reduced and irregular or lost cristae. Lastly, we have performed structural modeling of SMYD1 containing the p.Asn101Ser variant (N101S) and report how this variant may affect the enzymatic activity of SMYD1 due to its proximity to the substrate binding site. The identification of this novel variant constitutes the third patient with a SMYD1 variant displaying cardiomyopathy and provides insights into the molecular functionality of this protein. In addition, this is the first analysis of tissue from a patient expressing a SMYD1 variant which provides critical insights into the role of SMYD1 in the heart and how loss of function mutations can effect cardiac physiology.

Inhibition of de novo Fatty-Acid Biosynthesis in Isolated Chloroplasts by the Antibiotic Cerulenin and Its Synthetic Derivatives

1992 ◽  
Vol 47 (5-6) ◽  
pp. 382-386 ◽  
Author(s):  
Bernd List ◽  
Andrea Golz ◽  
Wilhelm Boland ◽  
Hartmut K. Lichtenthaler
Keyword(s):  
Fatty Acid ◽  
Inhibitory Activity ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Specific Inhibitor ◽  
Hydrocarbon Chain ◽  
Acetate Incorporation ◽  
Acid Biosynthesis ◽  
Structural Element ◽  
Isolated Chloroplasts

The antibiotic cerulenin was shown to be a potent dose-dependent inhibitor of de novo fattyacid biosynthesis in intact isolated chloroplasts of different plants (measured as [14C]acetate incorporation into the total fatty-acid fraction). Various chemical derivatives of cerulenin were synthesized and tested in the chloroplast assay-system of oat, spinach and pea. Modifications of the hydrocarbon chain of cerulenin (e.g. tetrahydro-cerulenin and its short-chain cis-2,3-epoxy-4-oxoheptanamide derivative) decreased the inhibitory activity of cerulenin, whereas variations of the epoxy-oxo-amide structural element led to a complete loss of inhibition potency. The results indicate that the naturally occurring antibiotic cerulenin is the most active specific inhibitor of de novo fatty-acid biosynthesis, but the formation of the hydroxylactam ring seems to be an essential requirement for the inhibitory activity. Those structural analogues of cerulenin, which can no longer form a hydroxylactam ring, do not possess any inhibitory capacity.

AN IMPROVED IN VITRO BIOASSAY METHOD FOR MEASURING LUTEINIZING HORMONE (LH) ACTIVITY USING MOUSE LEYDIG CELL PREPARATIONS

1974 ◽  
Vol 77 (4) ◽  
pp. 655-671 ◽  
Author(s):  
M.-P. Van Damme ◽  
D. M. Robertson ◽  
E. Diczfalusy
Keyword(s):  
Luteinizing Hormone ◽  
Leydig Cell ◽  
Marked Reduction ◽  
Improved Method ◽  
In Vitro Bioassay ◽  
Bioassay Method ◽  
Adult Mice ◽  
Assay Design ◽  
Mouse Leydig Cell

ABSTRACT An improved in vitro bioassay method for the measurement of LH activity is presented. The method is based on the assay of testosterone produced by "Leydig cell" preparations from mouse testes in the presence of added gonadotrophin. The method is significantly improved in terms of sensitivity, precision and practicability when compared to the previously described bioassay method employing decapsulated testes from adult mice. The sensitivity of the improved method is 15 μIU for HCG and 50 μIU for HMG. The useful range of the method is 15–260 μIU for HCG and 50–900 μIU for HMG. Using a 3 + 3 point assay design with each dose in quadruplicate, a mean index of precision (λ̅) of 0.044 was obtained in 19 assays. Human FSH, TSH, ACTH, LTH, STH, oxytocin, vasopressin and LHRH preparations did not influence the bioassay method at levels likely to be found in biological samples. A good correlation was found between estimates obtained by the "Leydig cell" method and by the method using decapsulated testes when various HCG and HMG preparations were used. With the proposed method at least 30 samples can be assayed each week by 2 persons, with a marked reduction in cost.

ACETATE AND CHOLESTEROL METABOLISM IN THE HUMAN FOETO-PLACENTAL UNIT AT MIDGESTATION

1970 ◽  
Vol 63 (1) ◽  
pp. 119-133 ◽  
Author(s):  
G. Telegdy ◽  
J. W. Weeks ◽  
D. F. Archer ◽  
N. Wiqvist ◽  
E. Diczfalusy
Keyword(s):  
Sodium Acetate ◽  
De Novo ◽  
Cholesterol Metabolism ◽  
Human Foetus ◽  
Homogeneous Form ◽  
Per Se ◽  
Hydroxy Progesterone

ABSTRACT Two complete foeto-placental units were perfused at midgestation with 5.0 mCi of uniformly labelled 14C-sodium acetate plus 5.0 mCi of cholesterol-7α-3H and two isolated foetuses were perfused with 2.5 mCi of 14C-labelled sodium acetate plus 2.5 mCi of cholesterol-7α-3H. The perfusions were carried out at 35–36°C for 90 min. The foetal adrenals, livers, testes and perfusates were analyzed and various labelled steroids were isolated in a radiochemically homogeneous form. Major quantities of 14C- and 3H-labelled pregnenolone (3β-hydroxy-pregn-5-en-20-one) and dehydroepiandrosterone (3β-hydroxy-androst-5-en-17-one) were isolated from the foetal perfusates, livers and adrenals. Minor amounts of 14C- and 3H-labelled progesterone (pregn-4-ene-3,20-dione) were isolated from the perfusates and adrenals, and 17α-hydroxy-progesterone (17α-hydroxy-pregn-4-ene-3,20-dione) and androstenedione (androst-4-ene-3,17-dione) from the perfusates. Smaller amounts of exclusively 14C-labelled androstenedione were isolated also from the adrenals. No labelled progesterone, androstenedione or testosterone (17β-hydroxy-androst-4-en-3-one) was detected in the testes. Attempts to isolate 20α-dihydroprogesterone (20α-hydroxy-pregn-4-en-3-one) or 20β-dihydroprogesterone (20β-hydroxy-pregn-4-en-3-one) from the perfusates, adrenals and livers failed. No labelled cortisol (11β,17,21-trihydroxy-pregn-4-ene-3,20-dione) or corticosterone (11β,21-dihydroxy-pregn-4-ene-3,20-dione) was detected in the adrenals, and no testosterone, oestrone (3-hydroxy-oestra-1,3,5(10)-trien-17-one) or 16α-hydroxy-dehydroepiandrosterone (3β,16α-dihydroxy-androst-5-en-17-one) in the perfusates. Pregnenolone and dehydroepiandrosterone (which were present in approximately equal amounts) accounted for as much as 95% of all steroids isolated from all sources. The bulk of these two steroids was isolated from the perfusates. The 14C/3H ratio of the pregnenolone and dehydroepiandrosterone isolated from the conjugated fraction of the perfusates was much higher than that of the unconjugated compounds. It is concluded that the midgestation human foetus is capable per se of carrying out the de novo synthesis of major quantities of pregnenolone and dehydroepiandrosterone. It is suggested that in foetal steroidogenesis acetate is utilized predominantly via a conjugated pathway and circulating cholesterol mainly via an unconjugated pathway.

scholarly journals Retinoic Acid Stimulates 17β-Estradiol and Testosterone Synthesis in Rat Hippocampal Slice Cultures

Endocrinology ◽  
2009 ◽  
Vol 150 (9) ◽  
pp. 4260-4269 ◽  
Author(s):  
Eiji Munetsuna ◽  
Yasushi Hojo ◽  
Minoru Hattori ◽  
Hirotaka Ishii ◽  
Suguru Kawato ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
De Novo ◽  
Fold Increase ◽  
Retinoid X Receptor ◽  
Rat Hippocampus ◽  
Male Rats ◽  
Receptor Signaling ◽  
Retinoic Acids ◽  
17Β Estradiol ◽  
Testosterone Synthesis

Abstract The hippocampus is essentially involved in learning and memory processes. Its functions are affected by various neuromodulators, including 17β-estradiol, testosterone, and retinoid. Brain-synthesized steroid hormones act as autocrine and paracrine modulators. The regulatory mechanism underlying brain steroidogenesis has not been fully elucidated. Synthesis of sex steroids in the gonads is stimulated by retinoic acids. Therefore, we examined the effects of retinoic acids on estradiol and testosterone biosynthesis in the rat hippocampus. We used cultured hippocampal slices from 10- to 12-d-old male rats to investigate de novo steroidogenesis. The infant rat hippocampus possesses mRNAs for steroidogenic enzymes and retinoid receptors. Slices were used after 24 h of preculture to obtain maximal steroidogenic activity because steroidogenesis in cultured slices decreases with time. The mRNA levels for P45017α, P450 aromatase and estrogen receptor-β in the slices were increased by treatment with 9-cis-retinoic acid but not by all-trans-isomer. The magnitude of stimulation and the shape of the dose-response curve for the mRNA level for P45017α were similar to those for cellular retinoid binding protein type 2, the transcription of which is activated by retinoid X receptor signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in the protein content of P45017α and a 2-fold increase in de novo synthesis of 17β-estradiol and testosterone. These steroids may be synthesized from a steroid precursor(s), such as pregnenolone or other steroids, or from cholesterol, as so-called neurosteroids. The stimulation of estradiol and testosterone synthesis by 9-cis-retinoic acid might be caused by activation of P45017α transcription via retinoid X receptor signaling.

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